Under anaerobic growth conditions, Escherichia coli produces hydrogenase and this enzyme activity is involved in H2 evolution (formate hydrogenlyase; FHL) and dihydrogen uptake. These metabolic processes are catalyzed by three hydrogenase isoenzymes. Besides the structural genes for the HYD isoenzymes, E. coli requires the products of several other genes for production of active hydrogenase. The fnr gene product and cAMP regulate H2 uptake. Studies on the regulation of hydrogenase indicate that the synthesis of active FHL requires besides the structural genes (fdhF and hyc), formate, molybdate as well as the products of fh1A and fh1B genes. Molybdate is a component of formate dehydrogenase (FDH- H) and thus is needed for activity. However, both fdhF and hyc operons coding for FDH-H and hydrogenase isoenzyme 3, respectively, are not transcribed in a molybdate-deficient medium. Besides these, the sigma54 subunit of RNA polymerase and DNA gyrase are also implicated in the H2 metabolism indicating the complexity of these metabolic processes. The long-term objective of this proposed study is to utilize H2 metabolism as a model anaerobic system, to study the regulation of anaerobic processes in a living cell. The specific objective of this proposal is to investigate the mechanism by which the cell regulates the transcription of the genes coding for the components of FHL in response to molybdate availability. The proposed experiments once completed will provide the following information. 1) The nature of control exerted by molybdate on FHL production. 2) Identification of a regulatory protein which senses the presence of molybdate in the cell. 3) The biochemical properties of this regulatory protein and its molybdate-binding characteristics. 4) The genetic and biochemical interaction of this protein with the upstream regulatory sequences of the FHL genes.